Application of BCL2 gene in stem cell-derived organoid 3D cluster culture

Abstract

This invention belongs to the field of organoid culture technology, specifically involving BCL2 Application of genes in 3D cluster culture of stem cell-derived organoids. Starting on day 4 of bovine embryonic stem cell cluster culture, the cell cluster edges became indistinct, cells began to die and darken, and the cell clusters began to diffuse. By day 7, the cell cluster survival rate was only 14.2%. This invention utilizes the overexpression of genes in bovine embryonic stem cells... BCL2 Following gene expression, the cells were cultured in clusters using the same method. The overexpressed cell clusters showed clear edges, and their volume increased continuously with the number of culture days. Even on day 7, clear cell cluster edges were still observable, and no diffuse cell death was observed; the survival rate remained high at 85.54%. BCL2 Genes can improve the survival rate of early cultured cell clusters of organoids, which is of great significance for the normal morphogenesis and functional maturation of organoids and can greatly improve the efficiency of organoid preparation.

Description

Technical Field

[0001] This invention belongs to the field of organoid culture technology, specifically involving BCL2 Application of genes in the 3D cluster culture of stem cell-derived organoids. Background Technology

[0002] Organoids, as three-dimensional cell culture models that mimic the structure and function of organs in vivo, have demonstrated significant application value in developmental biology, disease modeling, drug screening, and regenerative medicine. However, the in vitro construction of organoids typically requires a long-term culture process of at least 15 days, placing high demands on the stability of the culture system and the initial state of the cells. In the early stages of three-dimensional culture, the efficiency and survival rate of cell cluster formation directly determine the subsequent developmental process of the organoid. A low cell cluster formation and survival rate not only significantly reduces experimental reproducibility but also severely interferes with key biological processes such as cell proliferation, differentiation, and self-assembly, thereby affecting the normal morphogenesis and functional maturation of organoids and hindering subsequent research on related mechanisms and experimental applications. Therefore, improving the efficiency and survival rate of cell cluster formation in the early stages of organoid culture is of great significance for achieving stable and efficient long-term in vitro culture of organoids.

[0003] Bovine embryonic stem cells (bESCs) are pluripotent stem cells derived from the inner cell mass of early bovine embryos, possessing unlimited self-renewal capacity and the potential to differentiate into tissues of all three germ layers. Although studies have indicated that bovine embryonic stem cells are very similar to human embryonic stem cells in morphology and the expression of key pluripotency markers (such as OCT4), there is a significant gap in technological development between the two, and basic research on bovine embryonic stem cells has long lagged behind. Bovine embryonic stem cells have been found to possess unique metabolic characteristics different from human and mouse embryonic stem cells, and exhibit dual developmental plasticity in differentiating into both embryonic and extraembryonic tissues. The unique biological characteristics of bovine embryonic stem cells mean that their culture systems cannot be directly applied based on experience, thus posing greater challenges. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art, improve the survival rate of early cultured cell clusters of organoids, and make the obtained cell clusters have high clustering activity. The high survival rate can be well maintained until the seventh day, which is beneficial to subsequent research on organoids.

[0005] This invention provides BCL2 The application of genes in early-stage organoid cluster culture, the aforementioned BCL2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1; The early organoid culture refers to the cluster culture in the early stage of organoid culture.

[0006] Preferably, the BCL2Genes are used to improve cell clustering activity and / or cell cluster survival rate in early organoid culture.

[0007] Preferably, the clustering culture is a 3D clustering culture; The cell cluster survival rate is the cell cluster survival rate after 5-7 days of culture in clusters. The basic cells for the early culture of the organoids are embryonic stem cells.

[0008] This invention provides a method for improving the clustering activity and / or survival rate of embryonic stem cell clusters in culture, comprising the following steps: overexpressing [a specific ingredient] in embryonic stem cells. BCL2 Genes are then cultured in clusters. BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0009] Preferably, the agglomeration culture method is a hanging drop culture.

[0010] Preferably, the hanging drop culture step includes: seeding the culture medium containing embryonic stem cells into the inside of the culture dish lid at a rate of 25~30 μL / drop, and then flipping the culture dish lid over and placing it on the culture dish trough; the culture dish trough contains PBS.

[0011] Preferably, the time for the grouping culture is 5 to 7 days.

[0012] This invention provides BCL2 The application of the gene or the method described above in the preparation of organoids, wherein BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0013] Preferably, the organoid is a mammary gland organoid.

[0014] Preferably, the mammary gland organoid is a bovine mammary gland organoid, and the basic cells are bovine embryonic stem cells.

[0015] Beneficial effects: This invention describes the culture of bovine embryonic stem cells in clusters, revealing that they form well-defined, spherical cell clusters on days 0-3. However, starting on day 4, the cell cluster edges become less distinct, cells die and darken, and the clusters begin to diffuse. Ultimately, by day 7, only 14.2% of the cell clusters survive. To improve cell cluster culture efficiency, bovine embryonic stem cells are overexpressed with... BCL2 Following gene expression, the cells were cultured in clusters using the same method. The overexpressed cell clusters showed clear edges, and their volume increased continuously with the number of culture days. Even on day 7, clear cell cluster edges were still observable, and no diffuse cell death was observed. The cell cluster survival rate remained high at 85.54% on day 7. BCL2 Genes can improve the clustering activity and survival rate of early-stage organoid culture cells, which is of great significance for the normal morphological formation and functional maturation of organoids and can greatly improve the efficiency of organoid preparation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 A schematic diagram of 3D clustered hanging drop culture of bovine embryonic stem cells; Figure 2 An example of 3D clustered hanging drop culture of bovine embryonic stem cells; Figure 3 This is a microscopic image of 3D clustering of bovine embryonic stem cells cultured using conventional methods for different number of days in Example 1; the scale bar is 500 μm. Figure 4 For those carrying EGFP BCL2 A schematic diagram of overexpression of lentiviral plasmid; Figure 5 For bovine embryonic stem cells (bESC) and BCL2 Overexpression of embryonic stem cells ( BCL2 qPCR detection results of -OE); express P <0.05; Figure 6 Microscopic images of 3D clustering of bovine embryonic stem cells cultured by conventional methods for different number of days in the control and treatment groups of Example 1; scale bar is 500 μm. Figure 7 The results of immunofluorescence staining of mammary gland marker proteins in organoid cell clusters after 7 days of induction in the control and treatment groups of Example 1 are shown; the scale bar is 200 μm. Detailed Implementation

[0018] This invention provides BCL2 The application of genes in early-stage organoid cluster culture, the aforementioned BCL2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1.

[0019] As one implementation method, the present invention described BCL2Genes are used to improve cell clustering activity and / or cell cluster survival rate in early organoid culture. As one embodiment, the cell clustering culture described in this invention is 3D cell clustering culture. As one embodiment, the cell cluster survival rate described in this invention is the cell cluster survival rate after 5-7 days of culture. As one embodiment, the organoids described in this invention include mammary gland organoids. As one embodiment, the mammary gland organoids of this invention are bovine mammary gland organoids. As one embodiment, the basal cells for early organoid culture of this invention are embryonic stem cells. As one embodiment, the embryonic stem cells described in this invention are bovine embryonic stem cells. As one embodiment, the... BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0020] SEQ ID NO:1:MAHAGGTGYDNREIVMKYIHYKLSQRGYEWDAGDAGAAPPGAAPAPGILSSQPGRTPAPSRTSPPPPPAAAAGPAPSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLT PFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDSIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKALLSLALVGACITLGAYLGHK.

[0021] SEQ ID NO:2:

[0022] This invention provides a method for improving the survival rate of embryonic stem cell clusters in culture, comprising the following steps: overexpressing [a specific ingredient] in embryonic stem cells. BCL2 Genes are then cultured in clusters. BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0023] This invention relates to the overexpression of [a specific substance] in embryonic stem cells. BCL2There are no strict requirements regarding the genetic method; any conventional method in the field can be used, such as constructing genes with... BCL2 The gene is lentiviral vector, which is then introduced into embryonic stem cells to achieve... BCL2 Gene overexpression. This invention does not specify the type of culture medium; conventional embryonic stem cell culture medium, such as N2B27 medium, is sufficient. In one embodiment, the N2B27 culture medium comprises 40-50 mL DF12, 40-50 mL Neurobasal, 300-600 μL N2, 0.5-1 mL B27, 0.5-1 mL L-glutamine, 0.5-1 mL penicillin-dextrin antibiotics, 0.5-1 mL NEAA, and 50-100 μL β-mercaptoethanol. In another embodiment, the N2B27 culture medium comprises 48 mL DF12, 48 mL Neurobasal, 500 μL N2, 1 mL B27, 1 mL L-glutamine, 1 mL penicillin-dextrin antibiotics, 1 mL NEAA, and 100 μL β-mercaptoethanol. In one embodiment, the bispecific antibody described in this invention is a penicillin-streptomycin bispecific antibody, with a penicillin concentration of 10000 U / mL and a streptomycin concentration of 10000 µg / mL.

[0024] In one embodiment, the present invention digests and centrifuges bovine embryonic stem cells with mouse feeder layer, resuspends the stem cells in culture medium, and then re-spreads the cell suspension back into the culture dish and lets it stand for 30 min to 1 h to allow the mouse feeder layer to adhere to the bottom of the dish and the embryonic stem cells to be suspended in the culture medium. The culture medium is then recovered and thoroughly blown to disperse any undispersed cell clusters, thus obtaining bovine embryonic stem cells with mouse feeder layer removed.

[0025] In one embodiment, the cluster culture method described in this invention is a hanging drop culture. In one embodiment, the hanging drop culture steps of this invention include: seeding the culture medium containing embryonic stem cells at a rate of 25-30 μL / drop onto the inside of the culture dish lid, and then inverting the culture dish lid onto the culture dish inlet; the culture dish inlet contains PBS. In one embodiment, the cluster culture time of this invention is 5-7 days. This invention uses a hanging drop culture method where the liquid does not drip and creates a three-dimensional suspension environment for cell culture. In a specific embodiment of this invention, the amount of culture medium containing embryonic stem cells added is determined based on the desired number of organoid cell clusters, the concentration of the culture medium containing embryonic stem cells, and the concentration of each drop of culture medium containing embryonic stem cells. For example, the concentration of the culture medium containing embryonic stem cells is 10... 6 If the concentration of embryonic stem cells per drop is 250 cells / mL, then to culture 100 organoid cell clusters, the required culture medium concentration would be 250 cells / drop × 100 cells = 2.5 × 10⁻⁶ cells / mL. 4If there are 10 cells, then 10 cells need to be taken. 6 cells / mL ÷ 2.5 × 10 4 After counting the cells (40 μL per cell), centrifuge the suspension, discard the N2B27 culture medium, and then resuspend the cells in 2.5 mL of organoid culture medium containing 15% FBS-N2B27 (25 μL / drop × 100 cells). Finally, perform hanging drop culture at 25 μL / drop on the plate lid.

[0026] As one embodiment, the culture medium of the present invention can be N2B27 culture medium. The composition of N2B27 culture medium has been described above and will not be repeated here.

[0027] This invention provides BCL2 The application of the gene or the method described above in the preparation of organoids, wherein BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:1.

[0028] In one embodiment, the organoids of this invention include mammary gland organoids. In another embodiment, the organoids of this invention include bovine mammary gland organoids, with bovine embryonic stem cells as the basal cells. Using the method described in this invention, the resulting cell clusters exhibit more diverse morphologies, not simply spherical cell clusters. This not only recreates the morphology and biological characteristics of the mammary gland but also effectively reveals the formation process of the mammary gland during embryonic development, successfully cultivating mammary gland organoids. After directed differentiation, the expression levels of neurocadherin (N-Ca) decrease, while the expression levels of non-neurocadherin (E-Ca) increase. The expression levels of epithelial marker proteins TBX1, SIX1, and LEF1 also significantly increase.

[0029] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, explains the invention. BCL2 The application of genes in the 3D cluster culture of stem cell-derived organoids is described in detail, but it should not be construed as limiting the scope of protection of this invention.

[0030] Example 1 Three-dimensional (3D) cluster culture of bovine embryonic stem cells N2B27 culture medium was prepared according to the following ratio: 48 mL DF12, 48 mL Neurobasal, 500 μL N2, 1 mL B27, 1 mL L-glutamine, 1 mL penicillin (10000 U / mL) and streptomycin (10000 µg / mL), 1 mL NEAA, and 100 μL β-mercaptoethanol. 15% fetal bovine serum was added to the N2B27 culture medium to obtain organoid culture medium. Bovine embryonic stem cells were then cultured using a traditional cell clustering method for 3D culture. The specific steps are as follows: (1) Following existing technology (Yang L, Di AQ, Song LS, et al. Generation of modified cows and sheep from spermatid-like haploid embryonic stem cells[J]. Nature Biotechnology, 2025. https: / / doi.org / 10.1038 / s41587-025-02832-4), the purchased bovine embryonic stem cells were cultured with a mouse feeder layer to obtain bovine embryonic stem cells with a mouse feeder layer. Using a feeder layer removal kit (purchased from Miltenyi, product number 130-095-531), the bovine embryonic stem cells with the mouse feeder layer were digested and centrifuged, and the stem cells were resuspended in organoid culture medium. The cell suspension was then re-layered into the culture dish and allowed to stand for 30 min to 1 h. At this time, the mouse feeder layer adhered to the bottom of the dish, and the embryonic stem cells were still suspended in the culture medium. (2) The culture medium was recovered and thoroughly blown to disperse any undispersed cell clusters, and then cell counting was performed. (3) When performing 3D cluster culture, use the hanging drop culture method. Add the culture medium containing cells at a rate of 25 μL / drop, with each hanging drop containing approximately 250 cells, to the inside of the dish lid. Then, invert the dish lid and place it on the bottom of the dish containing PBS to prevent excessive evaporation of the liquid. Figure 1 and Figure 2 The results showed that a well-defined, spherical cell cluster could form during days 0-3 of culture. However, starting on day 4, the edges of the cell cluster became less distinct, cells began to die and darken, and the cell cluster began to diffuse. Ultimately, by day 7, only 14.2% of the cell cluster was still viable. Figure 3 As shown in Table 1.

[0031] Table 1. Survival rate of bovine embryonic stem cells cultured using traditional methods for different number of days.

[0032] Example 2 1. Construction of lentiviral plasmids Using pLVX-EF1α-IRES-Puro as the backbone vector, bovine cells labeled with EGFP green fluorescence were inserted between its EcoRI and SpeI sites. BCL2 Genes, and in cattle BCL2 A segment of EGFP, a fluorescent protein labeled with green, was added to the posterior end of the gene to obtain a recombinant vector containing EGFP. BCL2 The overexpression lentiviral plasmid is designated pLVX-EF1α-IRES-Puro-EGFP- BCL2 The diagram is as follows Figure 4As shown.

[0033] 2. Cow BCL2 overexpression of genes Referring to existing techniques (Huang Gaoxiang, Wei Dongmei, Yang Yao, et al. Construction of LILRA2 overexpression lentiviral vector and establishment of stable transfected cell line of THP-1-LILRA2[J]. Basic Medicine and Clinical, 2026, 46 (4): 498-503. DOI:10.16352 / j.issn.1001-6325.2026.04.0498.), bovine embryonic stem cells were overexpressed with bovine lentiviral vector. BCL2 Genes were selected, and cells exhibiting green fluorescence were identified as positive cells.

[0034] 3. BCL2 Overexpression verification Bovine embryonic stem cells and positive cells were collected separately, cultured for 7 days, and then detected by RT-qPCR. BCL2 The overexpression level; among which, BCL2 The upstream primer sequence for detection is: GGCGATGACTTCTCTCGGCG, SEQ ID NO:3; BCL2 The downstream primer sequence for detection is: GACAGCATCGCCCTGTGGA, SEQ ID NO:4.

[0035] The results showed that overexpression BCL2 Embryonic stem cells of genes ( BCL2 -OE) BCL2 The expression level was more than 30 times that of wild-type bovine embryonic stem cells (bESC), proving that... BCL2 Successful overexpression was achieved in bovine embryonic stem cells. Figure 5 ).

[0036] 4. Validation of three-dimensional (3D) cluster culture and clustering efficiency of bovine embryonic stem cells. Take conventional bovine embryonic stem cells (control group) and overexpressing BCL2 Embryonic stem cells containing the gene (treatment group) were cultured in clusters according to the conventional cell clustering method described in Example 1. During the clustering process, microscopic observation showed that the treatment group overexpressed the gene. BCL2 The cell clusters showed clear edges and increased in volume with increasing culture time; furthermore, clear cell cluster edges were still observed on day 7, and diffuse cell death was not observed. Figure 6 The cell cluster survival rate on day 7 was still as high as 85.54%, while the cell cluster survival rate of the control group was only 13.97% on day 7 (Table 2).

[0037] Table 2. Survival rate of bovine embryonic stem cells cultured using traditional methods for different number of days.

[0038] Example 3 Directional induction culture Take over expression BCL2 Embryonic stem cells containing the gene were randomly divided into a control group and a treatment group, and were treated as follows: (1) Differentiation of embryonic stem cell clusters into ectoderm: The first induction solution was prepared by adding 50 ng / mL BMP4, 1 μM SB431542, 25 ng / mL FGF2, and 1 μM LDN to organoid culture medium (N2B27 medium + 15% fetal bovine serum). The overexpressing group was then resuspended in the first induction solution. BCL2 Embryonic stem cells were cultured using a hanging drop method. The culture medium containing cells was added at a rate of 25 μL / drop, with approximately 250 cells per drop, onto the inside of a dish lid. The lid was then inverted and placed on the bottom of a dish containing PBS to prevent excessive evaporation. This was used for the first induction culture at 37°C and 5% CO2. The control group did not receive any targeted induction molecules and was cultured using organoid culture medium. (2) Differentiation of ectoderm into non-neuroectoderm: 25 ng / mL FGF2, 25 ng / mL FGF10, 10 μg / mL Wnt3a, 10 μg / mL Wnt5a, and 10 μg / mL Wnt10b were added to the organoid culture medium to obtain the second induction solution. The second induction solution was dispensed into the inside of the dish lid at a rate of 25 μL / drop. The embryonic stem cell clusters after the first induction culture (1) for 5 days were aspirated directly into the second induction solution using a pipette. The dish lid was then inverted and placed on the bottom of the dish containing PBS to prevent excessive evaporation of the liquid. The second induction culture was then performed for 1-5 days at 37°C and 5% CO2. The control group was cultured in organoid culture medium without the addition of directional induction molecules.

[0039] Ectodermal cells were cultured for 2 days to differentiate into non-neuroectoderm, i.e., 7 days of directed induction. Organoid cell clusters obtained were then analyzed for breast-related marker proteins using immunofluorescence staining. Results are as follows: Figure 7 As shown, the expression levels of neurocadherin (N-Ca) decreased and the expression levels of non-neurocadherin (E-Ca) increased after directed differentiation in the treatment group, demonstrating that the organoid cell clusters induced in the treatment group differentiated into non-neuroectoderm cells. The expression levels of epithelial marker proteins TBX1, SIX1, and LEF1 also significantly increased in the treatment group, indicating that the directed differentiated cell clusters differentiated into epithelial cells. Compared with the control group, the cell clusters obtained after directed induction culture in the treatment group exhibited more diverse morphologies and were not simply spherical cell clusters.

[0040] As can be seen from the above, overexpression BCL2 Genes can improve the survival rate of early cultured cell clusters of organoids, enabling organoids formed from embryonic stem cells to have high clustering activity. Their high survival rate can be well maintained until the seventh day, laying the foundation for the establishment of functional organoids.

[0041] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. BCL2 The application of genes in early-stage organoid cluster culture, the aforementioned BCL2 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:1; The BCL2 Genes are used to improve cell clustering activity and / or cell cluster survival rate in early organoid culture. The group formation cultivation is 3D group formation cultivation; The basic cells used in the early-stage cluster culture of the organoids were bovine embryonic stem cells.

2. The application according to claim 1, characterized in that, The cell cluster survival rate is the cell cluster survival rate after 5-7 days of culture.

3. A method for improving the clustering activity and / or survival rate of bovine embryonic stem cell cluster culture, characterized in that, The steps include: overexpression in bovine embryonic stem cells. BCL2 Genes are then cultured in clusters. BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

4. The method according to claim 3, characterized in that, The cluster culture method is suspended drop culture.

5. The method according to claim 4, characterized in that, The hanging drop culture step includes: seeding the culture medium containing embryonic stem cells into the inside of the culture dish lid at a rate of 25~30μL / drop, and then flipping the culture dish lid over and placing it on the culture dish trough; the culture dish trough contains PBS.

6. The method according to any one of claims 3 to 5, characterized in that, The time for group cultivation is 5 to 7 days.

7. BCL2 The use of the gene or the method according to any one of claims 3 to 6 in the preparation of bovine organs, wherein... BCL2 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

8. The application according to claim 7, characterized in that, The organoid is a mammary gland organoid.

Images